The present invention relates to retroviral vectors and their use in gene therapy. In particular it relates to complement resistant lentiviral vectors. The invention provides novel lentiviral packaging vectors, stable packaging cell lines, stable producer cell lines, recombinant retroviruses carrying a foreign gene of interest and methods relating to the use thereof, as well as methods for producing recombinant lentivirus in mammalian cells.
Retroviral vectors are a common tool for gene delivery (Miller, 1992, Nature 357: 455-460). The biology of retroviral proliferation enables such a use. Typically, wild type full length retroviral mRNAs serve both as a template for synthesis of viral proteins and as the viral genome. Such mRNAs encompass a region called the encapsidation signal which binds certain viral proteins thereby ensuring specific association of that mRNA with the produced virions. On infection of the target cell, reverse transcription of the retroviral mRNA into double stranded proviral DNA occurs. The retroviral enzyme, integrase, then binds to both long terminal repeats (LTR) which flank the proviral DNA and subsequently catalyzes the integration thereof into the genomic DNA of the target cell. Integrated proviral DNA serves as the template for generation of new full-length retroviral mRNAs.
Retroviral vectors have been tested and found to be suitable delivery vehicles for the stable introduction of a variety of genes of interest into the genomic DNA of a broad range of target cells. The ability of retroviral vectors to deliver unrearranged, single copy transgenes into a broad range of cells makes retroviral vectors well suited for transferring genes into cells. Unfortunately, in vivo use of retroviral gene delivery systems has been hindered by the inactivation of retroviruses by human serum. This inactivation may occur via complement-mediated lyses of retroviruses.
Attempts to protect gene transfer vectors from complement inactivation involve the co-administration of a soluble complement regulatory protein (CRP), or integration of a CRP into the viral membrane. One strategy has been used with murine leukemia virus, a retroviral delivery system, but requires the fusion of the catalytic domain of a CRP to a retroviral envelope protein in order to achieve incorporation of the CRP into viral particles (Spitzer et al., 1999, Human Gene Therapy 10:1893-1902). Another strategy has been the incorporation of a CRP into the baculovirus envelope to create a complement-resistant baculoviral vector for gene transfer (Hxc3xcser et al., 2001, Nature Biotechnology 19:451-455; WO 00/77233). This strategy also requires fusion of the CRP catalytic domain to the baculoviral envelope protein.
There remains a need for viral gene delivery systems that avoid immune responses directed against the transgene and resist complement inactivation without requiring the creation of a chimeric protein fusing CRP to a transmembrane region or the use of a separately administered product.
The invention disclosed herein offers a surprisingly feasible and efficient system for combining the advantages of retroviral gene delivery with complement resistance. The invention provides a lentiviral packaging system that comprises at least two vectors: a first vector comprises a first nucleotide sequence comprising a gag, a pol, or gag and pol genes; and a second vector comprises a second nucleotide sequence comprising a heterologous envelope gene and a gene that encodes a complement regulatory protein (CRP). In an alternative and preferred embodiment, the packaging system comprises at least three vectors, wherein the second nucleotide sequence comprises the envelope gene and not the CRP, and the gene encoding a CRP is provided on a third vector.
Preferably, the heterologous envelope gene comprises a VSV-G or baculoviral gp64 envelope gene. In a preferred embodiment, the lentivirus is HIV. A second or third generation packaging system is preferred, wherein the vectors lack a functional tat gene and/or functional accessory genes (vif, vpr, vpu, vpx, nef). In another preferred embodiment, the system further comprises an additional nucleotide sequence that comprises a rev gene. The packaging system can be provided in the form of a set of lentiviral vectors, or in the form of a packaging cell that contains the first, second, and, optionally, third nucleotide sequences. Preferably, the genes encoded by the nucleotide sequences are stably integrated into the genome of the packaging cell.
The invention further provides a producer cell that comprises the packaging system of the invention and a transgene. The producer cell of the invention is capable of producing a recombinant lentivirus that carries a transgene and resists complement inactivation. The recombinant lentivirus is capable of infecting a host cell, thereby delivering the transgene to the host cell for expression.
The invention additionally provides a method of delivering a transgene to a cell. The method comprises contacting the cell with a recombinant lentivirus of the invention. In one embodiment, the transgene is a therapeutic transgene. The cell can be in vivo, in vitro or ex vivo.